IGF-1 (insulin-like growth factor 1) is a hormone your body makes that tells muscle, bone, and other tissue to grow. Scientists discovered that if you snip three building blocks (amino acids) off the front end of IGF-1, the result - des(1-3)IGF-1, often just called "IGF-1 DES" - becomes 3 to 10 times more powerful in cell and animal experiments. That extra strength comes from the fact that it slips past the normal "chaperone" proteins in blood that usually hold IGF-1 back. Because of this potency, it has been studied for decades as a lab tool to understand growth, muscle wasting, and diabetes - and more recently it has shown up as an unregulated black-market product marketed to bodybuilders, despite never being approved or tested in people.
How strong is the evidence?
Every one of the 40 papers reviewed is either a lab dish (cell culture) experiment, an animal study (mostly rats, plus mice, pigs, and marmoset monkeys), or a technical paper about detecting the substance or manufacturing it. A few studies use human-derived cells or human tissue in a dish (prostate cells, colon cancer cells, liver cells, cartilage cells) - these are still lab experiments, not treatment of a living person. A 1996 review of this compound states plainly that 'clinical opportunities for des(1-3)IGF-I have not yet been evaluated,' and nothing in the following three decades of research changes that. There is no human dosing trial, no human safety trial, and no human outcomes data anywhere in this literature.
Uses
What people use it for
Bodybuilding and muscle-building (black market use)
AnecdotalIGF-1 DES is sold online, unregulated, as a research chemical marketed for building muscle and burning fat. This use is based entirely on animal data showing it builds lean tissue and blocks muscle breakdown - it has never been tested for this purpose in a real person, and it's on the World Anti-Doping Agency's banned list.
Research into muscle wasting and recovery from illness or injury
Animal / labIn lab animals, this compound has been used to study how the body might fight back against muscle loss from things like kidney failure, gut surgery, or steroid use. It is a research tool in these studies, not a treatment given to sick people.
Diabetes and blood sugar research
Animal / labAnimal studies use IGF-1 DES to understand how growth hormone-related hormones affect blood sugar, sometimes as a comparison or add-on to insulin. This is laboratory science, not a diabetes treatment that has reached people.
Basic brain and nerve research
Animal / labBecause IGF-1 plays a role in brain development and learning, researchers have used the DES version in rat brain-slice experiments to study how it affects nerve signaling. This is early-stage lab science with no connection to any human brain or cognitive benefit.
Potential benefits
What it may help with
Fights muscle breakdown in sick or catabolic animals
Animal / labIn rats with reduced kidney function, rats recovering from major gut surgery, and rats given steroid drugs that cause muscle wasting, IGF-1 DES consistently reduced the rate of muscle protein breakdown and improved nitrogen balance (a marker of the body holding onto, rather than losing, muscle protein). It usually outperformed regular IGF-1 at the same dose. All of this is in rats, not people.
Improves muscle protein building in diabetic animals
Animal / labIn diabetic rats, IGF-1 DES increased body weight, nitrogen retention, and muscle protein synthesis, working about as well as (or slightly better than) regular IGF-1, and reaching roughly 70% of the effect seen with insulin - though it did not fix the underlying high blood sugar or sugar in the urine.
Studies:1710892Lowers blood sugar more strongly and for longer than regular IGF-1
Animal / labIn pigs and marmoset monkeys, IGF-1 DES was 2 to 3 times more potent than regular IGF-1 at dropping blood sugar, and the effect lasted 4 to 8 times longer. Researchers noted this could theoretically make it useful alongside insulin therapy, but this idea has never been tested in a person and carries an obvious risk (see side effects).
Studies:9415072Boosts growth in growth-hormone-deficient mice
Animal / labIn a strain of dwarf mice that can't respond normally to growth hormone, daily injections of IGF-1 DES increased body length more than regular IGF-1 did, at a much lower dose.
Studies:2280209Possible nerve-signaling and brain-protective effects
Animal / labIn slices of rat brain tissue, IGF-1 DES boosted the strength of nerve-cell signaling in a brain region tied to learning and memory. In diabetic rats, it also reduced early warning-sign changes in the retina (the light-sensing part of the eye) that are linked to diabetic eye disease, even though it didn't fix the underlying high blood sugar. Both findings are lab/animal only.
Speeds up wound healing and cartilage growth in lab dishes
Animal / labIn cell-culture experiments, IGF-1 DES helped skin cells contract and close a wound-like gap in a collagen gel, and stimulated growth of human cartilage cells. These are early, dish-based findings and don't confirm a real healing benefit in living tissue.
What to watch for
Side effects & risks
- Serious
Dangerous, long-lasting low blood sugar (hypoglycemia)
Because IGF-1 DES is so much more potent at lowering blood sugar than regular IGF-1, and its effect lasts far longer, it carries a real risk of prolonged, hard-to-reverse hypoglycemia. This risk is confirmed in pigs and monkeys, and low blood sugar is a well-known danger of any IGF-1-type compound in humans - it just hasn't been formally tested in people for this specific version.
- Serious
Encourages tumor growth in animal studies
Mice bred to overexpress IGF-1 DES in mammary (breast) tissue developed far more breast tumors than normal mice, and the effect was worse when combined with a cancer-related gene mutation. Separately, in lab dishes, it stimulated growth of prostate cells from men with benign prostate enlargement. Because IGF-1 signaling is a known growth driver for several cancer types, this is treated as a serious theoretical risk, not just a lab curiosity.
- Moderate
Unwanted organ growth
In rat and mouse dosing studies, animals given IGF-1 DES showed increased weight of organs like the kidneys, heart, liver, lungs, stomach, and thymus - a sign the hormone drives growth throughout the body, not just in muscle. This mirrors known side effects of high-dose IGF-1 and growth hormone therapy in humans, like organ enlargement and joint or nerve swelling.
- Serious
No human safety testing exists
There is no published data on how IGF-1 DES behaves in a real person - not dosing, not side effects, not interactions. Anyone using it is relying entirely on rat, mouse, pig, and monkey data plus general knowledge about how IGF-1 behaves in humans.
- Moderate
Unverified black-market products
A drug-testing lab found that products sold on the black market as IGF-1 DES for bodybuilding often contained degraded, lower-quality peptide - meaning what's in the vial may not match the label.
Dosing
Dosing — what studies used
There is no established or approved human dose for IGF-1 DES - it has never been given to a person in a published study. Every dose below comes from animal research, mostly rats, using injections under the skin (often via a small implanted pump) or into muscle over periods of days to a few weeks. These numbers describe what scientists gave lab animals to study a specific biological question, not a treatment plan for a person. Anyone using an unregulated version of this peptide is doing so with zero clinical dosing guidance.
Growth-hormone-deficient (dwarf) mice, growth study
Animal study3 or 30 micrograms per animal
Once daily · 3 weeks · Injection (site not specified)
Even the low, 3-microgram dose outperformed a 30-microgram dose of regular IGF-1, showing the compound's extra potency in animals.
Rats with reduced kidney function (catabolic/wasting model)
Animal study0.9 mg per kg of body weight per day
Continuous infusion · 7 days · Subcutaneous, via implanted osmotic pump
Compared against regular IGF-1 at 0.9 and 2.2 mg/kg/day; the DES version matched or beat the higher IGF-1 dose on some measures at a lower amount.
Rats recovering from major gut (bowel) surgery
Animal study0.96 mg per kg of body weight per day
Continuous infusion · 7 days · Subcutaneous, via implanted osmotic pump
Produced weight gain and improved nitrogen balance similar to a nearly 2.5x higher dose of plain IGF-I.
Diabetic rats, muscle and metabolism study
Animal studyAbout 1.08 mg per kg of body weight per day
Continuous infusion · 7 days · Subcutaneous, via implanted osmotic pump
Effects on nitrogen balance were roughly 70% as strong as high-dose insulin, but blood sugar itself was not corrected.
Blood sugar-lowering potency study
Animal study20 to 270 micrograms per kg of body weight, single dose
Single bolus injection · Effects tracked for 4 hours · Bolus injection (pigs and marmoset monkeys)
Used to compare glucose-lowering strength and duration against plain IGF-1 and insulin; not a repeat-dosing protocol.
Anti-doping detection study
Animal study100 micrograms per kg of body weight
Single dose · Detectable in blood up to 24 hours afterward · Intramuscular injection (rats)
This study was about detecting the substance for drug testing, not about establishing an effective or safe dose.
Because it disappears from the bloodstream quickly and has no established human dose, any real-world use is essentially guesswork on amount, frequency, and duration - stacked on top of the fact that it has never been safety-tested in a person at any dose.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
Your body already makes IGF-1, a hormone that tells muscle and other tissue to grow. Normally, most of the IGF-1 in your blood gets grabbed and held by special "carrier" proteins (called binding proteins), which act like a leash, keeping most of it from reaching your cells at any given moment. IGF-1 DES has three building blocks clipped off its front end, and that small change stops the carrier proteins from grabbing onto it nearly as well. With less of a leash holding it back, more of it stays free to bind directly to growth receptors on cells - which is why it acts several times stronger than regular IGF-1 in lab and animal experiments. The tradeoff is that this same freedom from carrier proteins means it also gets broken down and cleared out of the blood much faster.
Who should avoid it
- Anyone with a personal or family history of cancer, especially breast or prostate cancer - animal data shows this compound can speed up tumor growth
- People with diabetes or anyone prone to low blood sugar - the blood-sugar-lowering effect is strong, prolonged, and unpredictable without medical monitoring
- Pregnant or breastfeeding people - no safety data exists at all
- Children and teens - no safety data, and growth-hormone-axis compounds carry particular risk in still-developing bodies
- Competitive athletes - it is banned by the World Anti-Doping Agency and can be detected in testing
- Anyone without medical supervision - there is no approved product, no verified dosing, and no safety monitoring available outside a research setting
- Anyone relying on an unregulated online seller - lab testing has found degraded, lower-quality product sold under this name
Interactions to know
- Insulin and other diabetes medications - IGF-1 DES lowers blood sugar strongly and for a long time in animal studies; combined with insulin or other glucose-lowering drugs, the risk of dangerous low blood sugar would likely be higher, though this hasn't been tested in people
- Growth hormone or other IGF-1-type products - stacking multiple growth-hormone-axis compounds has not been studied and could compound both the growth effects and the risks
- Any cancer treatment or condition - because it may promote tissue growth broadly, combining it with cancer therapy or having an active cancer diagnosis raises theoretical concerns
The papers that matter most
Key studies
A 1996 review summarizing what this compound is, why it's roughly 10 times more potent than regular IGF-1 (it avoids being held back by blood carrier proteins), and stating explicitly that no clinical (human) testing had been done - a statement still true decades later.
Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I.
Shows IGF-1 DES drops blood sugar 2-3 times more powerfully than regular IGF-1, with an effect lasting up to 8 times longer - the clearest evidence of both a potential use and a serious safety risk.
IGF-I variants which bind poorly to IGF-binding proteins show more potent and prolonged hypoglycaemic action than native IGF-I in pigs and marmoset monkeys.
Mice engineered to overexpress IGF-1 DES in breast tissue developed significantly more mammary tumors, and this worsened when combined with a cancer-related gene mutation - the key cancer-risk signal in this evidence set.
Cooperative interaction between mutant p53 and des(1-3)IGF-I accelerates mammary tumorigenesis.
One of the clearest muscle-building/anti-wasting demonstrations, with real dosing data, but also shows it didn't fix the underlying diabetes - reinforcing that this is a growth-promoting compound, not a blood sugar treatment.
Increased weight gain, nitrogen retention and muscle protein synthesis following treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1-3)IGF-I.
Explains why this compound is both more potent and faster-clearing than regular IGF-1: it isn't held onto by blood carrier proteins, so more of it reaches tissue, but it also disappears from the blood roughly 3-4 times faster.
Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I in rats.
Confirms this compound is a banned substance in sport, sold on the black market for bodybuilding, and that black-market products often contain degraded, lower-quality peptide.
Detection of LongR3-IGF-I, Des(1-3)-IGF-I, and R3-IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes.
Bottom line
IGF-1 DES is a genuinely fascinating lab discovery - a tweak to natural IGF-1 that makes it several times more powerful in animals - but it exists purely as a research tool and black-market product, not a tested treatment. The animal data hints at real muscle-preserving and blood sugar effects, alongside real warning signs for cancer risk and dangerous low blood sugar. With zero human trials of any kind, anyone using it is experimenting on themselves with a substance that has never been checked for human safety or dosing.
Research papers
Studies we have on file for IGF-1 DES. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
40 papers
Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I.
Des(1-3)IGF-I, a truncated variant of human IGF-I with the tripeptide Gly-Pro-Glu absent from the N-terminus, has been isolated from bovine colostrum, human brain and porcine uterus. This protein probably results from post-translational cleavage of IGF-I. Des(1-3)IGF-I generally is about 10-fold more potent than IGF-I at stimulating hypertrophy and proliferation of cultured cells, a consequence of much reduced binding to IGF-binding proteins, in turn caused by the absence of the glutamate at position 3. The increased potency is retained in part when the variant is administered in vivo, with selective anabolic effects particularly evident in gut tissues. Clinical opportunities for des(1-3)IGF-I have not yet been evaluated, but could apply in catabolic states as well as for the treatment of inflammatory bowel diseases.
Enzymatic conversion of IGF-I to des(1-3)IGF-I in rat serum and tissues: a further potential site of growth hormone regulation of IGF-I action.
We recently identified and characterized a protease present in rat serum which is capable of generating des(1-3)IGF-I. In this study, we have investigated the effects of GH deficiency and replacement on the activity of this protease in rat serum and tissue extracts. Protease activity was significantly higher in sera from hypophysectomized (hypox) rats than sham-operated rats (P < 0.001) and GH treatment of hypox rats (human GH, 100 micrograms/100 g body weight i.p. for 10 days) significantly reduced the levels towards normal. The addition of IGF-I to hypox rat serum to achieve IGF-I concentrations comparable with or greater than that seen in normal rat serum had no effect on the measured protease activity. Protease activity was also detected in tissue extracts. The level of protease activity in the various tissues from sham-operated rats demonstrated the following order: liver > testes > heart > skeletal muscle > lung > thymus > kidney > brain > spleen. In all tissue extracts examined, except that from the lung, the levels of protease activity were higher in extracts from hypox rats compared with sham-operated rats. The largest differences between tissue extracts from hypox and sham-operated rats were seen in spleen (4-fold higher), kidney (2.27-fold), testes (1.55-fold) and heart (1.31-fold). In the liver, kidney and testes, GH treatment significantly reduced protease activity. Since the pattern of serum IGF-binding proteins (IGFBPs) differ in hypox rats compared with normal rats, we determined whether these changes could result in enhanced serum binding of des(1-3)IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF analogue, on growth hormone and IGF-binding protein secretion from cultured rat anterior pituitary cells.
The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I, a potent IGF-I analogue, on the secretion of GH and IGF-binding protein (IGFBP) from cultured rat anterior pituitary cells were measured. IGF-I and des(1-3)IGF-I stimulated GH secretion at low concentrations (maximally effective at 1 and 0.1 micrograms/l respectively) and inhibited GH secretion at higher concentrations. The half-maximal inhibitory concentrations (IC50) were approximately 20 micrograms/l and 1 microgram/l for IGF-I and des(1-3)IGF-I respectively. Thus des(1-3)IGF-I was more potent than IGF-I in these effects on GH secretion. We postulate that the increased potency of des(1-3)IGF-I in affecting GH secretion is due to decreased binding of this peptide by pituitary IGFBP compared with IGF-I. In contrast with IGF-I and des(1-3)IGF-I, IGF-II did not stimulate GH secretion at low concentrations, but did inhibit GH secretion from pituitary cells with an IC50 of approximately 20 micrograms/l. Several IGFBPs ranging in molecular mass from 22,000 to 52,000 were detected in medium conditioned by cultured anterior pituitary cells. When measured by Western-ligand blotting and competitive ligand-binding techniques, these IGFBPs exhibited decreased binding of des(1-3)IGF-I compared with IGF-I and IGF-II. The production of IGFBP by anterior pituitary cells was stimulated by the addition of IGFs to the culture medium.
Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1-3) IGF-I in Chinese hamster ovary cells.
Recombinant human insulin-like growth factor-I (hIGF-I) and a biologically potent variant lacking the N-terminal tripeptide (des(1-3)IGF-I) were produced from transfected Chinese hamster ovary cells. The constructs encoding the signal peptide, sequence of the mature peptide and a C-terminal extension peptide were expressed under the control of a Rous sarcoma virus promoter. Successfully transfected clones secreting correctly processed recombinant hIGF-I or des(1-3)IGF-I were selected by their secretion of IGF-I-like activity into the culture medium. The recombinant peptides were purified to homogeneity as assessed by high-performance liquid chromatography and N-terminal sequence analysis. The purified recombinant peptides exhibited biological potencies equivalent to authentic IGF-I and des(1-3)IGF-I respectively.
Des (1-3) IGF-I potently enhances differentiated cell growth in olfactory bulb organ culture.
We recently provided evidence that newborn rat olfactory bulb (OB) could be maintained in serum-free organ culture with combinations of insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF), both of which are locally synthesized. Des (1-3), or truncated, IGF-I is a potent analog of IGF-I isolated from rat and human brain. We proposed in this study to examine the effects of des (1-3) IGF-I on cell function, morphology and on neuronal and glial cell differentiation in our cultured OB model, using cell-specific immunostains for neurons (150 kDa neurofilament) and glial cells (glial fibrillary associated protein--GFAP). OB were cultured in Iscove's serum-free medium containing IGF-I or des (1-3) IGF-I both alone or in combination with bFGF. Dose dependent responses of 14C amino acid uptake showed des (1-3) IGF-I to be 3-5 fold more potent than IGF-I with a half maximal response at about 20 ng/ml in comparison to 100 ng/ml of IGF-I. The maximum response to IGF-I +/- bFGF was seen at 150 ng/ml; a ten-fold higher dose of insulin +/- bFGF was required to achieve the same response. While morphology was close to fresh 6 day OB following culture with IGF-I (150 ng/ml) and bFGF (25 ng/ml), the substitution of des (1-3) IGF-I at 50 ng/ml markedly improved morphology. Neurons were identified following culture in IGF-I or bFGF alone, but showed greater organisation in the mitral layer following combined IGF-I/bFGF culture. However, in contrast to IGF-I (150 ng/ml), des (1-3) IGF-I (50 ng/ml) supported marked neuronal expression. Furthermore, when des (1-3) IGF-I (50 ng/ml) was substituted for IGF-I, in combination with bFGF, the pattern of enhanced neuronal expression in the mitral layer was very close to that seen in the fresh 6 day bulb, with dendrites projecting to the glomerular layer. In OBs treated with no growth factors, or either IGF-I, des (1-3) IGF-I or bFGF alone, glial expression was widespread and poorly organised, suggesting an injury response. In contrast, following treatment with combinations of bFGF with IGF-I or des (1-3) IGF-I, a more ordered, though enhanced glial response was seen in glomerular and granule cell layers.(ABSTRACT TRUNCATED AT 400 WORDS)
Enhanced potency of truncated insulin-like growth factor-I (des(1-3)IGF-I) relative to IGF-I in lit/lit mice.
The relative potencies of insulin-like growth factor (IGF-I) and the N-terminal truncated derivative, des(1-3)IGF-I, have been compared in lit/lit mice. Injection of 30 micrograms IGF-I, 30 micrograms des(1-3)IGF-I or 3 micrograms des(1-3)IGF-I daily for 3 weeks increased total length and nose-rump length of the animals substantially more than in controls or animals treated with 3 micrograms IGF-I daily. Body weight changes were not statistically significant. The lower dose of des(1-3)IGF-I, but not that of IGF-I, led to increases in kidney and heart weights relative to controls, while the higher dose of either IGF-I or des(1-3)IGF-I also increased the weights of liver, lungs and stomach. These results indicate that the higher potency of des(1-3)IGF-I demonstrated in cultured cells also applies in vivo to at least one strain of GH-deficient animals.
Des (1-3) IGF-I-stimulated growth of human stromal BPH cells is inhibited by a vitamin D3 analogue.
Prostate growth and differentiation is under the control of androgens not only during fetal life and childhood but also in adulthood, and it has been proposed that increased prostatic concentration of androgens, or increased androgen responsiveness, causes benign prostatic hyperplasia (BPH). However, different androgen ablation strategies such as treatment with GnRH agonists and finasteride resulted in a modest decrease of the hyperplastic prostate volume. In the last few years it became evident that both androgen-dependent and androgen-independent growth factors promote prostate enlargement by inducing cell proliferation or reducing apoptosis. Therefore, new therapeutic strategies, aimed at reducing intraprostatic growth factor signaling, are under investigation. In this study, we report further evidence that a non hypercalcemic-analogue of vitamin D(3), analogue (V) decreases growth factor-induced human BPH cell proliferation and survival. We found that Des (1-3) insulin-like growth factor [Des (1-3) IGF-I], an IGF-I analogue, which does not bind to IGF-binding proteins, is a potent mitogen for BPH stromal cells via a dual mechanism: stimulation of cell growth and inhibition of apoptosis. Similar results were previously reported for another growth factor for BPH cells, keratinocyte growth factor (KGF). Accordingly, we speculate that both KGF and IGF might be involved in the pathogenesis of BPH. We also found analogue (V) not only inhibits the mitogenic activity of growth factors on BPH cells, but even decreased the basal expression of bcl-2, and induced apoptosis. Therefore, vitamin D(3) analogues might be considered for the medical treatment of BPH.
IGF-I and its variant, des-(1-3)IGF-I, enhance growth in rats with reduced renal mass.
The efficacy of insulin-like growth factor I (IGF-I) in enhancing growth in animals with reduced renal mass was investigated in subtotally nephrectomized young male rats. Recombinant human IGF-I was administered by osmotic minipumps for 7 days at two doses, 0.9 and 2.2 mg.kg body wt-1. day-1, and the truncated analogue of IGF-I, des-(1-3)IGF-I, was given at a dose of 0.9 mg.kg body wt-1.day-1. The partial nephrectomy procedure resulted in significantly impaired renal function as evidenced by elevated serum urea and creatinine concentrations, reduced creatinine clearance, and increased average daily urine output. Carcass composition was significantly altered in animals with reduced renal mass; water content increased and fat content decreased, while protein content remained unchanged. Carcass composition was not affected by IGF treatment. Body weight gain, food utilization, and nitrogen balance during the treatment period were significantly increased in rats treated with IGF-I at both the lower and higher doses and in those treated with des-(1-3)IGF-I. The improved nitrogen balance in the des-(1-3)IGF-I group could at least partly be explained by a diminished rate of muscle protein breakdown, as indicated by the reduced urinary excretion rate of 3-methylhistidine. Compensatory hypertrophy of the remnant kidney was significantly increased in the group treated with the high dose of IGF-I. These results suggest that IGF-I may have beneficial effects on somatic growth and nitrogen balance in renal insufficiency, with des-(1--3)IGF-I being particularly effective in reducing the rate of muscle protein breakdown.
Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I in rats.
Incubation of 125I-labelled insulin-like growth factor-I (IGF-I) with rat plasma at 4 degrees C led to the transfer of approximately half the radioactivity to 150 kDa and smaller complexes with IGF-binding proteins. The extent of association was greater with labelled IGF-II and essentially absent with the truncated IGF-I analogue, des(1-3)IGF-I. A greater degree of binding of IGF peptides with binding proteins occurred after i.v. injection of the tracers into rats, but most of the des(1-3)IGF-I radioactivity remained free. Measurement of the total plasma clearances showed the rapid removal of des(1-3)IGF-I compared with IGF-I and IGF-II; the mean clearances were 4.59, 1.20 and 1.34 ml/min per kg respectively. The mean steady-state volume of distribution was larger for des(1-3)IGF-I than for IGF-I and IGF-II (461, 167 and 181 ml/kg respectively), probably because of the differences in plasma protein binding. With all tracers, radioactivity appeared in the kidneys to a greater extent than in other organs. The amount of radioactivity found in the adrenals, brain, skin, stomach, duodenum, ileum plus jejunum and colon was in rank order, des(1-3)IGF-I greater than IGF-I greater than IGF-II. Since this ranking is the opposite of the abilities of the three IGF peptides to form complexes with plasma binding proteins, we propose that the plasma binding proteins inhibit the transfer of the growth factors to their tissue sites of action. Moreover, we suggest that IGF analogues that are cleared rapidly from blood may have greater biological potencies in vivo.
IGF-I and the truncated analogue des-(1-3)IGF-I enhance growth in rats after gut resection.
Effects of insulin-like growth factor I (IGF-I) administration and that of the truncated analogue des-(1-3)IGF-I have been examined in 170-g rats over a 7-day period after surgery to remove 80% of the jejunum plus ileum. The doses administered via osmotic infusion pumps were 0.96 and 2.4 mg.kg-1.day-1 IGF-I and 0.96 mg.kg-1.day-1 des-(1-3)IGF-I. All groups lost weight on the day after surgery, but over the next 3 days the des-(1-3)IGF-I and high-dose IGF-I groups stabilized better and subsequently gained significantly (P less than 0.05) more weight than the vehicle or low-dose IGF-I groups over the last 3 days. The weight gains (mean +/- SE) for the groups over this last 3-day period were 14.0 +/- 1.7, 14.4 +/- 2.9, 21.9 +/- 1.7, and 20.8 +/- 1.0 g for the vehicle, low-dose IGF-I, high-dose IGF-I, and des-(1-3)IGF-I groups, respectively. The nitrogen balances over the last 3 days for the high-dose IGF-I and des-(1-3)IGF-I groups, at 242 +/- 14 and 217 +/- 13 mg/d, respectively, were significantly (P less than 0.05) more positive than the control group at 153 +/- 21 mg/d. These differences could at least partially be explained by changes in muscle protein breakdown, as assessed by 3-methyl-L-histidine excretion. The kidneys were heavier in all treatment groups and the thymus after administration of des-(1-3)IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes.
Insulin-like growth factor-I (IGF-I) and its analogs LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I are prohibited substances in sport. Although they were never approved for use in humans, they are readily available as black market products for bodybuilding and can be used to enhance physical performance. This study's aims were to validate a fast and sensitive detection method for IGF-I analogs and to evaluate their detectability after intramuscular administration in rats. The sample preparation consisted of an immunopurification on MSIA™ microcolumns using a polyclonal anti-human-IGF-I antibody. The target substances were then directly analyzed by nano-liquid chromatography coupled with high-resolution mass spectrometry. Abundant signs of lower quality, oxidized peptide forms were found in black market products, justifying the need to monitor at least both the native and mono-oxidized forms. The analytical performance of this method (linearity, carry over, detection limits, precision, specificity, recovery, and matrix effect) was studied by spiking the analogs into human serum. Following a single intramuscular administration (100 μg/kg) in rats, detection was evaluated up to 36 h after injection. While unchanged Des(1-3)-IGF-I and R3 -IGF-I were detected until 24 h after administration, LongR3 -IGF-I disappeared rapidly after 4 h. Des(1)-LongR3 -IGF-I, a new N-terminal Long-R3 -IGF-I degradation product, was detected in addition to Des(1-10)-LongR3 -IGF-I and Des(1-11)-LongR3- IGF-I: the latter was detected up to 16 h. The same products were found after in vitro incubation of the analogs in human whole blood, suggesting that observations in rats may be extrapolated to humans and that the validated method may be applicable to antidoping testing.
des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.
HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.
Generation of des-(1-3) insulin-like growth factor-I in serum by an acid protease.
Des-(1-3) insulin-like growth factor-I (IGF-I), a more potent IGF-I variant, has been isolated from biological fluids and tissue extracts. The mechanisms involved in this posttranslational modification of IGF-I have not been elucidated. In this study we demonstrated the presence in rat serum of an acid protease activity that is capable of generating des-(1-3) IGF-I from intact IGF-I. An assay for measuring this enzyme activity using an immobilized synthetic substrate, biotin-G-P-E-T-L-C, that includes the site of cleavage of intact IGF-I was developed. Using this assay, the time, temperature, and pH dependency of the enzyme activity was investigated. Although some activity was observed under neutral conditions, the optimal pH was pH 5.5. Of a variety of commercially available proteases tested, only trypsin and cathepsin-D had activity. In contrast to the serum enzyme activity, trypsin was more active at pH 7.5 than at pH 5.5, and cathepsin-D resulted in extensive proteolysis of IGF-I. Soybean trypsin inhibitor, alpha 1-antitrypsin, and pepstatin-A were able to partially inhibit the serum enzyme activity. This enzymatic activity was partially purified from serum by acid-ethanol extraction (6-fold) and further purified by pepstatin-A affinity chromatography (200-fold). The partially purified enzyme had characteristics similar to those of the activity measured in serum. The enzymatic conversion of [125I]IGF-I to [125I]des-(1-3) IGF-I in serum was confirmed by immunoprecipitation using antibody to an N-terminal fragment of IGF-I (residues 1-9), which could recognize intact IGF-I, but not des-(1-3) IGF-I (< 3% cross-reactivity), and by size-exclusion HPLC analysis. When [125I]IGF-I was incubated with serum, only the 7.5-kilodalton free IGF-I fraction contained the des-(1-3) IGF-I variant. [125I]des-(1-3) IGF-I was not detected in the 150- to 200- or 50-kilodalton IGF-I-binding protein complexes. These findings demonstrate that des-(1-3) IGF-I can be generated in serum at neutral pH and that this process may be enhanced under the acidic conditions that occur in the pericellular environment.
Cooperative interaction between mutant p53 and des(1-3)IGF-I accelerates mammary tumorigenesis.
Mammary tumorigenesis was analysed in transgenic mice which overexpress des(1-3)hIGF-I (WAP-DES) and/or a mutant form of p53 (p53172R-H). Nonlactating, multiparous WAP-DES mice exhibited hyperplastic lesions termed mammary interepithelial neoplasia (MIN) which constitutively expressed WAP-DES. By 23 months of age, 53% of the WAP-DES mice developed mammary adenocarcinomas. A 75% reduction in both apoptosis and proliferation was observed in the normal mammary glands of WAP-DES mice. Mammary tumor incidence in WAP-DES/p53 bitransgenic mice was similar to that of WAP-DES and 2 - 3-fold greater than that of nontransgenic and p53172R-H females. Tumor latency, however, was reduced by 8 months in bitransgenic mice as compared to mice of the other three genotypes. Aneuploidy was frequently observed in tumors from bitransgenic and p53172R-H mice, but not from mice expressing only the WAP-DES transgene. Expression of IGFBP3 was elevated in tumors from WAP-DES, but not bitransgenic mice, indicating an alteration in the p53/IGF-I axis. These studies indicate that overexpression of des(1-3)hIGF-I increases the frequency of MIN and stochastic mammary tumors and that the appearance of tumors displaying genomic instability is accelerated by mutant p53172R-H. Oncogene (2000) 19, 889 - 898.
Immunoradiometric assay measurements of insulin-like growth factor-I (IGF-I): comparison with radioimmunoassays using native or des (1-3) IGF-I as radioligands.
Insulin-like growth-factor-binding proteins (BPs) in serum interfere with the measurement of insulin-like growth factor-I (IGF-I). Various assays have been developed to overcome this interference. We evaluated an immunoradiometric (IRMA) assay and compared it with the radioimmunoassay (RIA) using both native IGF-I and a truncated form of IGF-I [des (1-3) IGF-I] as radioligands. The IRMA was simpler (one step assay) and faster (3 hr incubation) than RIA(s) (overnight incubation). Sera were extracted with acid ethanol (AE) before all three assays. Analysis of serum samples (n = 78) performed by use of the two different radioligands in the RIA assays were highly correlated (r = 0.967, P < 0.0001). Measurements of serum IGF-I by IRMA in same samples were also highly correlated with those of the RIA assays (r = 0.952 for RIA and 0.947 for triGF-I RIA, P < 0.0001 for both). To assess the effect of binding protein-3 (BP-3) levels (after AE extraction) on these assays, BP-3 levels were measured in sera from 36 healthy women. The mean BP-3 level was 3.6 +/- 0.79 (S.D.) mg/L (range 1.3-5.0), and there was no significant difference in IGF-I levels measured by the three assays. Also, BP-3 levels were inversely correlated with IGF-I levels as measured by all three methods (r = 0.73 for IRMA, 0.71 for triGF-I, and 0.75 for IGF-I RIA). To assess the effect of binding protein-1 (BP-1) levels on these assays, IGF-I was also measured by IRMA and trlGF-I RIA in 19 women with advanced breast cancer. Women with breast cancer had significantly higher (P < 0.001) BP-1 levels than age matched healthy women. IGF-I levels measured by IGF-I IRMA were slightly lower than those measured by trlGF-I RIA in breast cancer patients. However, this difference was not statistically significant (P = 0.56). These findings suggest that variations in BP-3 or BP-1 levels after AE extraction have no significant effect in any of these assays. "We conclude that trlGF-I as a radioligand provides no added advantage over the standard IGF-I RIA. We also conclude that the IRMA assay is valid for measuring IGF-I and is faster and more convenient than RIA.
Des(1-3)IGF-1 treatment normalizes type 1 IGF receptor and phospho-Akt (Thr 308) immunoreactivity in predegenerative retina of diabetic rats.
Little is known about interventions that may prevent predegenerative changes in the diabetic retina. This study tested the hypothesis that immediate, systemic treatment with an insulin-like growth factor (IGF)-1 analog can prevent abnormal accumulations of type 1 IGF receptor, and phospho-Akt (Thr 308) immunoreactivity in predegenerative retinas of streptozotocin (STZ) diabetic rats. Type 1 IGF receptor immunoreactivity increased approximately 3-fold in both inner nuclear layer (INL) and ganglion cell layer (GCL) in retinas from STZ rats versus nondiabetic controls. Phospho-Akt (Thr 308) immunoreactivity increased 5-fold in GCL and 8-fold in INL of STZ rat retinas. In all cases, immunoreactive cells were significantly reduced in STZ des(1-3)IGF-1-treated versus STZ rats. Preliminary results suggested that vascular endothelial growth factor (VEGF) levels may also be reduced. Hyperglycemia/failure of weight gain in diabetic rats continued despite systemic des(1-3)IGF-1. These data show that an IGF-1 analog can prevent early retinal biochemical abnormalities implicated in the progression of diabetic retinopathy, despite ongoing hyperglycemia.
IGF-I stimulates chemotaxis of human neuroblasts. Involvement of type 1 IGF receptor, IGF binding proteins, phosphatidylinositol-3 kinase pathway and plasmin system.
SH-SY5Y human neuroblastoma cells express IGF receptors, IGFs and IGF binding proteins (IGFBPs), and provide a model for studying the role of the IGF system in human neuronal development. We investigated the effect of IGF-I and des(1-3)IGF-I on the motility of SH-SY5Y cells by a cell migration assay based on the assessment of the number of cells which migrated across 8 microm pore size membranes and around an agarose drop. IGF-I and des(1-3)IGF-I stimulated neuroblast chemotaxis in a dose-dependent manner. Treatment of cells with these agents for 24 h resulted in a significant increase (IGF-I by 70% and des(1-3)IGF-I by 90%; P<0. 0001) in cell motility relative to control conditions. Addition of monoclonal antibody against type 1 IGF receptor (alpha-IR3), significantly (P<0.05) reduced the cell motility induced by IGF-I (by 30%) and des(1-3)IGF-I (by 70%). Wortmannin, a specific inhibitor of phosphatidylinositol (PI)-3 kinase intracellular signalling, also reduced the IGF-stimulated cell migration (by over 40%, P<0.01), indicating a key role of the PI-3 kinase pathway in mediating the IGF effect on neuroblast migration. Finally, cell treatment with plasminogen (PLG) markedly enhanced neuroblast migration (by over 200%, P<0.01), whereas incubation with the PLG inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride reduced cell motility (by 80%, P<0.01), thus suggesting an involvement of PLG-dependent IGFBP proteolysis in the regulation of neuroblast motility. In conclusion, IGF-I is a potent stimulator of neuroblast migration through the activation of type 1 IGF receptor and the PI-3 kinase intracellular pathway. IGFBPs and the plasmin system seem to play a role in cell motility, although the nature and the extent of their involvement has yet to be elucidated.
Effects of chronic renal failure on plasma clearance of insulin-like growth factor I, des-(1-3)IGF-I, and LR3IGF-I.
Using a rat model of chronic renal failure (CRF), we examined insulin-like growth factor I (IGF-I) clearance, degradation, organ distribution, and IGF binding profiles in plasma. The effects of IGF-binding proteins (IGFBP) on IGF clearance and degradation in CRF were studied using the IGF-I analogues des-(1-3)IGF-I and LR3IGF-I, which bind poorly to IGFBP. Although total clearance of IGF-I was not significantly altered in CRF, half-life and area under the curve were increased in the rapid distribution phase and were reduced in the slow elimination phase. Total clearance of LR3IGF-I was significantly increased. Reduced binding of IGF-I in the 150-kDa complex and increased binding to smaller-molecular-weight IGFBP were observed in CRF. Increased degradation of both IGF-I and LR3IGF-I was associated with reduced IGF binding in the 150-kDa complex. The results suggest that the accumulation of lower-molecular-weight IGFBP with reduced IGF binding in the 150-kDa complex, associated with increased degradation of peptide, may explain, at least in part, the reduced bioactivity of IGF-I observed in CRF.
Insulin-like growth factor I and its variant, des(1-3)IGF-I, improve nitrogen balance and food utilization in rats with renal failure.
The growth-promoting effects of IGF-I were examined in rats following partial nephrectomy and compared with the effects of the des(1-3) variant of IGF-I. Four groups of rats were subjected to 5/6 nephrectomy (n = 8 per group) and treated for 7 days with IGF-I (0.9 or 2.2 mg/kg BW/day), des(1-3)IGF-I (0.9 mg/kg/BW/day), or vehicle (0.1 M acetic acid) administered subcutaneously by osmotic pump. A group of vehicle-treated, sham-operated control rats (n = 7) was included. Food utilization was significantly improved in all three peptide-treated groups, by 13-16% compared with the vehicle-treated nephrectomized group. Also, nitrogen balance was enhanced, particularly in the des(1-3)IGF-I group, in which nitrogen excretion was reduced by 24%, with the low- and high-dose IGF-I groups showing 16 and 18% reductions, respectively. Serum urea levels were significantly decreased, by 25%, in the des(1-3)IGF-I group, with 20 and 17% reductions being observed in the low- and high-dose IGF-I groups. Muscle protein degradation was found to be significantly attenuated with des(1-3)IGF-I treatment but was not significantly affected in the two IGF-I-treated groups. While carcass composition was not altered with IGF peptide treatment, absolute mass of protein in the carcass was improved in rats treated with the high dose of IGF-I. These results show that IGF-I or, more particularly, des(1-3)IGF-I may be efficacious in overcoming impaired growth in renal failure.
Insulin-like growth factor binding protein-1 from Hep G2 cells is potently inhibited by the truncated IGF-I analogue des-(1-3) IGF-I.
Des-(1-3) insulin-like growth factor-I (IGF-I) is an IGF analogue lacking aminoacid 1 to 3 which displays reduced binding to insulin-like growth factor binding protein-1 (IGFBP-1). A greater inhibition of immunoreactive IGFBP-1 was obtained with des-(1-3) IGF-I (10 ng/ml) in Hep G2 medium when incubated in Eagle's Modified Essential Medium (EMEM) without phenolred compared to EMEM with phenolred; EMEM without phenolred was chosen for further experiments. Des-(1-3)IGF-I decreases dose dependently the concentration of IGFBP-1, with a maximal effect at 3-10 micrograms/l when incubated for 24 h; 10 micrograms/l of des-(1-3)IGF-I caused a small but significant inhibition of IGFBP-1 after 8 h incubation and this inhibition was 41% and 33% of controls after 14 and 19 h incubation. The relative potencies at 16 h of incubation of IGF-I and insulin in suppressing IGFBP-1 in comparison to des-(1-3)IGF-I were 0.41 (0.25-0.78) and 0.08 (0.01-0.26), respectively. A dose-dependent decrease of IGFBP-1 mRNA to 30% of control was observed after 4 h incubation with 0.1-10 micrograms/l des-(1-3)IGF-I. Changes of glucose concentration (0-20 mmol/l) in the medium did not affect the IGFBP-1 concentration in the medium. In summary: Des-(1-3)IGF-I was tenfold more potent than insulin, and threefold more potent than IGF-I in decreasing IGFBP-1 concentration in medium conditioned by Hep G2 cells.
Role of IGF-I in normal mammary development.
Growth hormone (GH) is now believed to be the pituitary factor that is responsible for mammary ductal morphogenesis. Mammary development at puberty occurs because of synergy between GH and estrogen on formation of terminal end buds (TEBs). TEBs extend into the substance of the mammary gland fat pad, resulting in ductal morphogenesis. Ultimately, the whole mammary fat pad accommodates a complex network of ducts. IGF-I or des(1-3) IGF-I mimic the actions of GH on TEB formation in hypophysectomized, gonadectomized rats. Since GH stimulates IGF-I mRNA within the mammary gland synergistically, we hypothesize that IGF-I partially mediates actions of GH in mammary gland development. Studies in transgenic mice overexpressing IGF-I, des(1-3) IGF-I, or IGFBP-3 show that IGF-I causes ductal hypertrophy in the lactating mouse and prevention of post-lactational mammary gland involution. One of the mechanisms for this effect involves apoptosis. The potential role of GH or IGF-I in mammary carcinogenesis, and the applicability of animal studies to humans, are discussed.
Biodistribution of 125I-labeled des(1-3) insulin-like growth factor I in tumor-bearing nude mice and its in vitro catabolism.
Insulin-like growth factor I (IGF-I) is a potent mitogen for many tumor cell lines, and IGF-I receptors are overexpressed in many tumors. Specific IGF-binding proteins (IGFBPs) modulate the interaction of IGF and its receptors. Consequently, radiolabeled IGF-I has been considered for tumor imaging. In the present study, we investigated the biodistribution of 125I-labeled des(1-3)IGF-I, a truncated analogue of IGF-I, in tumor-bearing nude mice. Additional studies included its catabolism by tumor cells in vitro and its binding to serum IGFBPs in vivo in nude mice. We also compared groups that were and were not injected with unlabeled peptide analogue. Our data showed that 125I-labeled des(1-3)IGF-I catabolized very fast, with a rapid appearance of nonprecipitable iodine, when incubated at 37 degrees C, but it was not catabolized at 4 degrees C incubation. 125I-labeled des(1-3)IGF-I was bound to serum-binding proteins, mainly in a complex with a molecular weight of M(r) 150,000. The uptake of radioactivity in normal tissues decreased quickly with time, particularly in the kidneys. In mice receiving higher doses of des(1-3)IGF-I, the radioactivity in all normal tissues was lower than in the mice with no carrier-added des(1-3)IGF-I, except in the stomach and spleen. These data suggest that 125I-labeled des(1-3)IGF-I is rapidly internalized after binding to the IGF receptor and is rapidly catabolized with release of breakdown products. Lower specific activity of 125I-labeled des(1-3)IGF-I resulted in altered biodistribution, including faster blood clearance and higher tumor uptake, by decreasing the formation of complexes with IGFBPs.
Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells.
Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.
The role of the insulin-like growth factor system in the developing brain.
Insulin-like growth factors (IGFs) play a central role in brain growth and development, with IGF-1, its receptors and binding proteins (IGFBPs) being highly expressed, particularly in the olfactory bulb (OB), representing a local paracrine system. A potential role of IGFBPs in transporting and targeting IGFs to their receptors is supported by the finding that abundant IGFBP-2 is associated with cell surface proteoglycans. An OB organ culture system expressing IGF-1 and IGFBPs represents an in vitro injury model. Although IGF-1 is potently antiapoptotic for neurons and oligodendrocytes, marked gliosis and IGFBP-3 expression are seen, similar to in vivo injury responses, where intraventricular injection of IGF-1 postinjury enhances neuronal survival by blocking apoptosis. In contrast, des (1-3) IGF-1, which has low affinity for IGFBPs, is ineffective, supporting a transport or receptor targeting role for local IGFBPs. We conclude that the IGF system plays a crucial cell survival and growth role in the developing and injured brain and that IGFs have significant therapeutic potential.
Insulin-like growth factor (IGF)-binding proteins inhibit the biological activities of IGF-1 and IGF-2 but not des-(1-3)-IGF-1.
(1) Many cell types secrete insulin-like growth factor (IGF)-binding proteins that can be expected to sequester free IGF and modify the biological activities of the growth factors. (2) A binding protein purified from bovine kidney (MDBK) cells potently inhibited the ability of IGF-2 to stimulate DNA synthesis or protein accumulation as well as to reduce rates of protein breakdown in chick embryo fibroblasts. The binding protein did not influence the biological activities of des-(1-3)-IGF-1, while effects on IGF-1 were intermediate. Since the chick embryo fibroblasts contain only the type 1 IGF receptor, the MDBK-cell binding protein must have reduced the accessibility of IGF-2 and IGF-1 to that receptor. Binding to the type 2 receptor on L6 myoblasts was also inhibited. (3) Inhibiting effects on both protein breakdown responsiveness to IGF and IGF binding to cell receptors were also observed with human amniotic fluid binding protein, although here IGF-1 and IGF-2 were equipotent. These results contrast with stimulatory responses on different IGF-1 actions of the same binding protein reported previously [Elgin, Busby & Clemmons (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3254-3258]. (4) The biological potencies of IGF-1, IGF-2 and des-(1-3)-IGF-1 correlate inversely with their binding to proteins released into the medium by cells, so that the enhanced potency of des-(1-3)-IGF-1 is a consequence of it not binding to purified binding proteins or those released by cultured cells.
Increased weight gain, nitrogen retention and muscle protein synthesis following treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1-3)IGF-I.
We have examined the effects of infusing recombinant human growth hormone (hGH), insulin-like growth factor-I (IGF-I), the truncated IGF-I analogue, des(1-3)IGF-I, and insulin over a 7-day period in streptozotocin-induced diabetic rats. IGF-I at a dose of 1.05 or 1.08 mg/kg per day in two experiments increased body weight and nitrogen retention above those of vehicle-infused controls to about 30% of the improvement achieved with 25 or 30 units of insulin/kg per day, but only in the second experiment were the differences statistically significant (P less than 0.05). A 2.5-fold higher IGF-I dose, or des(1-3)IGF-I at 1.08 mg/kg per day, gave effects that were approx. 70% of those obtained with insulin. hGH at 1.38 mg/kg per day was not effective. The IGF peptides, unlike insulin, did not ameliorate the diabetic glucosuria. The improvements in nitrogen balance could be accounted for in part by increases in muscle protein synthesis. Muscle protein breakdown, as assessed by 3-methylhistidine excretion, was inhibited by insulin, but not by the IGF peptides. Carcass fat increased substantially following insulin administration. This did not occur with the IGF peptides, suggesting that IGF predominantly stimulates the growth of lean tissue. IGF-I concentrations and IGF-I-binding proteins in plasma were increased by IGF-I, especially at the higher dose, whereas hGH produced only a transient increase in IGF-I. Des(1-3)IGF-I induced binding proteins, but had only a slight effect on measured IGF-I concentrations. We conclude that IGF peptides stimulate muscle protein synthesis and improve nitrogen balance in diabetes without obviously influencing the abnormal carbohydrate metabolism. Moreover, des(1-3)IGF-I is at least as potent as the full-length IGF-I.
Involvement of growth factors in the regulation of pubertal mammary growth in cattle.
Pubertal mammary growth in heifers is dependent on interactions of many hormones and growth factors of which some are stimulatory while others are inhibitory. Although estrogen and growth hormone (GH) are of primary importance, more recent studies have suggested a role for both systemic and mammary tissue-specific growth factors. Growth factors may act as mediators of estrogen and GH or through specific effects of their own. These growth factors include insulin (INS), IGFs (IGF-I and IGF-II), epidermal growth factor (EGF), FGFs (FGF-1 and FGF-2), TGFs (TGF-alpha and TGF-beta's, amphiregulin (AR), platelets derived growth factor (PDGF), and mammary derived growth factor-1 (MDGF-1). Using mammary epithelial cells derived from prepubertal heifers and cultured in three-dimensional collagen gels as an in vitro model, we have investigated the mitogenic effects of a number of different growth factors (IGF-I, des(1-3) IGF-I, IGF-II, INS, EGF, TGF-alpha, AR, FGF-1, FGF-2, and TGF-beta 1). As expected, IGF-I, des(1-3)IGF-I, IGF-II and INS all stimulated proliferation of mammary cells with des(1-3)IGF-I being the most potent and INS the least potent. The mitogenic effect of IGF-I could be inhibited by both IGFBP-2 and IGFBP-3 showing that these binding proteins modulate the bioactivity of IGF-I in the mammary gland at the cellular level. Regulation of IGF availability by IGFBPs in the extracellular environment therefore is critical for IGF action in the mammary gland. Proliferation of mammary epithelial cells was also stimulated by growth factors of the EGF family, i.e. EGF, TGF-alpha and AR, however, not as much as growth factors from the IGF family. Members of the fibroblast growth factor family showed various mitogenic activities. FGF-1 stimulated DNA synthesis while FGF-2 in concentrations above 10 ng/ml inhibited DNA synthesis. TGF-beta 1 at very low concentrations stimulated proliferation slightly whereas higher concentrations strongly inhibited proliferation of mammary epithelial cells and inhibited mitogenesis induced by growth factors of both the EGF- and IGF family. This shows that TGF-beta 1 is a very potent regulator of pubertal mammary growth.
Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes.
Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.
Functional characterization of des-IGF-1 action at excitatory synapses in the CA1 region of rat hippocampus.
Insulin-like growth factor-1 (IGF-1) and growth hormone play a major role in the growth and development of tissues throughout the mammalian body. Plasma IGF-1 concentrations peak during puberty and decline with age. We have determined that chronic treatments to restore plasma IGF-1 concentrations to adult levels attenuate spatial learning deficits in aged rats, but little is known of the acute actions of IGF-1 in the brain. To this end, we utilized hippocampal slices from young Sprague-Dawley rats to characterize the acute effects of des-IGF-1 on excitatory synaptic transmission in the CA1 region. We observed a 40% increase in field excitatory postsynaptic potential (fEPSP) slope with application of des-IGF-1 (40 ng/ml) and used whole cell patch-clamp recordings to determine that this enhancement was due to a postsynaptic mechanism involving alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) but not N-methyl-D-aspartate receptors. Furthermore, the enhancement was completely blocked by the broad-spectrum tyrosine kinase inhibitor, genistein (220 microM), and significantly reduced by the PI3K blockers wortmannin (1 microM) and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (10 microM), suggesting that the effect was predominantly dependent on PI3K activation. This characterization of the acute actions of des-IGF-1 at hippocampal excitatory synapses may provide insight into the mechanism by which long-term increases in plasma IGF-1 impart cognitive benefits in aged rats. Increases in AMPA receptor-mediated synaptic transmission may contribute directly to cognitive improvement or initiate long-term changes in synthesis of proteins such as brain-derived neurotrophic factor that are important to learning and memory.
Efficient transdermal delivery of functional protein cargoes by a hydrophobic peptide MTD 1067.
The skin has a protective barrier against the external environment, making the transdermal delivery of active macromolecules very difficult. Cell-penetrating peptides (CPPs) have been accepted as useful delivery tools owing to their high transduction efficiency and low cytotoxicity. In this study, we evaluated the hydrophobic peptide, macromolecule transduction domain 1067 (MTD 1067) as a CPP for the transdermal delivery of protein cargoes of various sizes, including growth hormone-releasing hexapeptide-6 (GHRP-6), a truncated form of insulin-like growth factor-I (des(1-3)IGF-I), and platelet-derived growth factor BB (PDGF-BB). The MTD 1067-conjugated GHRP-6 (MTD-GHRP-6) was chemically synthesized, whereas the MTD 1067-conjugated des(1-3)IGF-I and PDGF-BB proteins (MTD-des(1-3)IGF-I and MTD-PDGF-BB) were generated as recombinant proteins. All the MTD 1067-conjugated cargoes exhibited biological activities identical or improved when compared to those of the original cargoes. The analysis of confocal microscopy images showed that MTD-GHRP-6, MTD-des(1-3)IGF-I, and MTD-PDGF-BB were detected at 4.4-, 18.8-, and 32.9-times higher levels in the dermis, respectively, compared to the control group without MTD. Furthermore, the MTD 1067-conjugated cargoes did not show cytotoxicity. Altogether, our data demonstrate the potential of MTD 1067 conjugation in developing functional macromolecules for cosmetics and drugs with enhanced transdermal permeability.
Combination of insulin-like growth factor (IGF)-I and IGF-binding protein-1 promotes fibroblast-embedded collagen gel contraction.
Wound contraction is an important event that minimizes the wound defect during the healing process. Involvement of insulin-like growth factor (IGF)-I and IGF-binding protein (IGFBP)-1 in wound contraction was studied using an in vitro model. Human dermal fibroblasts (1 x 10(5) cells/ml) were incorporated into a porcine type I collagen (0.21% final) in serum-free medium. The fibroblast-embedded collagen gels in a 12-well plate were floated from the well, and various reagents were then added to the assay medium. The surface area of the gel was calculated by measuring the diameters of the collagen gel. IGF-I at high doses (30 and 100 ng/ml) revealed 6.8% (P < 0.01) and 7.7% (P < 0.001) gel contraction, respectively, and des (1-3) IGF-I at 10 ng/ml produced a 4.5% gel contraction (P < 0.01). Meanwhile, IGFBP-I did not induce any significant contraction throughout the tested concentrations (0.1-100 ng/ml). A combination of IGF-I and IGFBP-1 at 1 ng/ml of each reagent, a concentration at which gel contraction was not observed when each of the reagents was tested individually, produced a 14% gel contraction (P < 0.001), whereas combinations of des (1-3) IGF-I with IGFBP-1 at the same concentrations did not promote gel contraction. The increased IGFBP-I doses in combination with 1 ng/ml IGF-I tended to enhance the gel contraction. IGF-I- and IGFBP-1-induced gel contraction was prominent during the initial 12-h incubation period. When anti-IGF-I, anti-IGFBP-1, or anti-IGF-I receptor antibody was added to the assay medium before the addition of IGF-I and IGFBP-1, the IGF-I- and IGFBP-1-induced gel contraction was significantly suppressed (P < 0.001). Endothelin-1, a vasoconstrictor peptide that is known to promote fibroblast-embedded collagen gel contraction, appeared to be partially involved in the IGF-I- and IGFBP-1-induced gel contraction, because the addition of an endothelin receptor antagonist (Bosentan or BE-18257B at 1 microg/ml) moderately suppressed the IGF-I- and IGFBP-1-induced gel contraction (P < 0.01). On the other hand, when IGF-I and IGFBP-1 were applied with endothelin-1 (1 nM), an enhanced gel contraction (29.4%) was observed that was significantly greater than that induced by either individually (P < 0.001). These results clearly indicate that the combination of IGF-I and IGFBP-1 promotes fibroblast contraction in collagen gel, and that this phenomenon is caused by IGFBP-1's strong potentiation of the IGF-I-induced gel contraction.
The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits the des (1-3) insulin-like growth factor-I generating protease.
The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action.
Effect of IGFBP-3 on IGF- and IGF-analogue-induced insulin-like growth factor-I receptor (IGFIR) signalling.
Insulin-like growth factor binding protein-3 (IGFBP-3) binds IGF-I and IGF-II with high affinity, at least an order of magnitude higher than the affiniy of the IGFs for the IGFIR. It has been hypothesized that IGFBP-3 inhibits IGF binding to the IGFIR via a mechanism independent of its ability to sequester IGFs. In the present study, we examined the effects of IGFBP-3 and its proteolytic fragments on the initial events of the IGFIR signalling pathway. IGFBP-3 inhibited IGF-I-, IGF-II-, Des(1-3)IGF-I- and Long(R3)IGF-I-induced IGFIR phosphorylation in a dose-dependent manner at similar concentration range but not QAYL-induced IGFIR-P. The((1-97))IGFBP-3 fragment was able to inhibit only IGF-I-induced IGFIR-P. The((1-97))IGFBP-3 fragment but not intact IGFBP-3 inhibited insulin-induced IGFIR-P. Monolayer cross-linking with [(125)I]IGFBP-3 indicated that there is no direct interaction of IGFBP-3 with the IGFIR. This study demonstrates that the effect on the initial step of IGFIR signalling by IGFBP-3 is largely due to its ability to sequester IGF and the IGF analogues in the extracellular milieu and not the result of any interaction of IGFBP-3 with the IGFIR or a mechanism independent of its ability to bind IGFs.
An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.
Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.
IGF-I variants which bind poorly to IGF-binding proteins show more potent and prolonged hypoglycaemic action than native IGF-I in pigs and marmoset monkeys.
The relative acute hypoglycaemic potencies of IGF-I and several variants of IGF-I which bind poorly to the IGF-I binding proteins (IGFBPs) have been examined in marmosets (Callithrix jacchus) and the pig. In the marmoset study, IGF-I and des(1-3)IGF-I were compared in anaesthetised and conscious animals in a range of bolus doses from 42 to 270 micrograms/kg body weight. In the pig study, IGF-I was compared with four variants, des(1-3)IGF-I long-IGF-I, R3IGF-I and long-R3IGF-I (LR3IGF-I), which show reduced affinity for the IGFBPs as well as with insulin. Doses in the pig were 20 and 50 micrograms/kg body weight for the IGFs and 3 micrograms/kg for insulin. In each study serial blood samples were taken from 30 min before to 4 h after the bolus injection. Plasma glucose levels were decreased in a dose-responsive manner with the pig more sensitive than either the conscious or anaesthetised marmoset (maximum lowering 4.8, 3.7 and 2.5 mmol/l respectively). The IGF variants were consistently 2- to 3-fold more potent than IGF-I in each animal for lowering of plasma glucose to the nadir, with the potency reflecting the relative affinities for binding to the IGFBPs and the IGF-I receptors. Thus, hypoglycaemic potency was in the order IGF-I < long-IGF-I < R3IGF-I approximately LR3IGF-I < des (1-3)IGF-I. Notably the variants suppressed plasma glucose levels over a much longer period than did IGF-I, the cumulative suppression over four hours showing an approximately 4- to 8-fold increase in the extent of hypoglycaemia. The prolonged suppression was not simply proportional to the hypoglycaemic nadir; at doses equipotent for glucose lowering, the cumulative hypoglycaemic effect for the variants in either species was about 2-fold that for IGF-I. The differential effect of the variants in the marmoset could not be accounted for by correlated changes in plasma insulin, IGF-I or IGFBP levels in plasma. Indirect effects via inhibition of glucagon, or direct effects via hepatic insulin receptors are postulated to account for the results. There was a dose-related reduction in plasma amino acids in the pig but, unlike the case for plasma glucose, only one analogue, LR3IGF-I was more potent than IGF-I. The response to LR3IGF-I was accentuated at the high dosage but on the basis of the other variants tested this effect could not be ascribed to either of the incorporated molecular variations. Despite their more rapid clearance from the circulation, variants of IGF-I which show lower affinity for binding to IGFBPs show proportionately superior potency for sustained hypoglycaemic action. Since our data were obtained in animal models of accepted relevance to humans these results point to the possible superior efficacy of the variants, especially des(1-3)IGF-I, over IGF-I for use as an adjunct to insulin treatment of hyperglycaemic conditions.
Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are anabolic in dexamethasone-treated rats.
The administration of insulin-like growth factor-I (IGF-I) via subcutaneously implanted osmotic pumps partially reversed a catabolic state produced by the co-administration of 20 micrograms of dexamethasone/day to 150 g male rats. Marked dose-dependent effects on body weight and nitrogen retention were produced, with the highest IGF-I dose, 695 micrograms/day, giving a 6 g increase in body weight over 7 days, compared with a 19 g loss in the dexamethasone-only group and an 18 g gain in pair-fed controls. Two IGF-I analogues that bind poorly to IGF-binding proteins, the truncated form, des(1-3)IGF-I, and a variant with an N-terminal extension as well as arginine at residue 3, LR3IGF-I, were approx. 2.5-fold more potent than IGF-I. The response with LR3IGF-I was particularly striking because this peptide binds 3-fold less well than IGF-I to the type 1 IGF receptor. The increased potencies of the IGF-I variants may relate to the substantially increased plasma levels of IGF-binding proteins, particularly IGFBP-3, produced by the combined treatment of dexamethasone with IGF-I or the variants. These binding proteins would be expected to decrease the transfer of IGF-I, but not that of the variants, from blood to tissue sites of action. Measurements of muscle protein synthesis at the end of the treatment period and muscle protein breakdown by 3-methylhistidine (3MH) excretion throughout the experiment indicated coordinate anabolic effects of the IGF peptides on both processes. Thus 3MH excretion was decreased at the highest IGF-I dose from 83.5 +/- 4.2 (S.E.M.) mumol/kg per 7 days to 65.1 +/- 2.2, compared with 54.9 +/- 1.2 in the pair-fed controls. Part of this response in 3MH excretion may have reflected a decrease in gut protein breakdown, because IGF-I and especially the IGF analogues increased the gut weight by up to 45%. Notwithstanding the effects on protein synthesis and breakdown, the fractional carcass weights remained low in the IGF-treated groups, although the increase in total carcass weight reflected nitrogen rather than fat gain. The dexamethasone-induced changes in liver, spleen and heart weight were restored towards normal by the IGF treatment. The experiment demonstrates the potential of IGF-I treatment of catabolic states and especially the value of modified forms of growth factors that bind weakly to IGF-binding proteins.
Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency.
An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
Interactions of IGF-1 with the blood-brain barrier in vivo and in situ.
Insulin-like growth factor-1 (IGF-1) given peripherally has been found effective in clinical trials to slow down neuronal degeneration in some nervous system diseases. This raises the question of whether and how IGF-1 crosses the blood-brain barrier (BBB). In this report, we found that IGF-1 had a half-life of 4.5 min in blood, could remain intact for 20 min, and entered brain and spinal cord linearly. In the brain, IGF-1 had an influx rate of 0.4 microl/g x min after intravenous (iv) bolus injection as determined by multiple-time regression analysis. Intact radiolabeled IGF-1 was present in brain at 20 min after iv injection. Most of the injected IGF-1 entered the brain parenchyma instead of being entrapped in the cerebral vasculature. Addition of nonradiolabeled IGF-1 enhanced the influx of radiolabeled IGF-1 after iv injection, but inhibited the influx of radiolabeled IGF-1 by in-situ brain perfusion, suggesting that protein binding can explain the difference between the iv and perfusion experiments. In the spinal cord, the cervical region had the fastest uptake, followed by lumbar spinal cord. The thoracic spinal cord had the slowest uptake, comparable to that of brain. By contrast, des(1-3)IGF-1, an IGF-1 analogue with little protein binding but similar biological activity, had a shorter half-life in blood, slower influx rate into brain, and no alteration in pharmacokinetics after addition of nonradiolabeled peptide. We conclude that IGF-1 enters the CNS by a saturable transport system at the BBB, which functions in synchrony with IGF binding proteins in the periphery to regulate the availability of IGF-1 to the CNS.
Insulin-like growth factor (IGF) binding protein-3 inhibits type 1 IGF receptor activation independently of its IGF binding affinity.
Insulin-like growth factor binding proteins (IGFBPs) regulate the cellular actions of the IGFs owing to their strong affinities, which are equal to or stronger than the affinity of the type 1 IGF receptor (IGF-IR), the mediator of IGF signal transduction. We recently found that IGFBP-3 modulates IGF-I binding to its receptor via a different mechanism possibly involving conformational alteration of the receptor. We have now investigated the effects of IGFBP-3 on the initial steps in the IGF signaling pathway. MCF-7 breast carcinoma cells were preincubated with increasing concentrations of IGFBP-3 and then stimulated with IGF-I, des(1-3)IGF-I, or [Q(3)A(4)Y(15)L(16)]-IGF-I, the latter two being IGF-I analogs with intact affinity for the type 1 IGF receptor, but weak or virtually no affinity for IGFBPs. Stimulation of autophosphorylation of the receptor and its tyrosine kinase activity was dose-dependently depressed. At 2.5 nM, IGFBP-3 provoked more than 50% inhibition of the stimulation induced by 3 nM des(1-3)IGF-1 and, at 10 nM, more than 80% inhibition. Similar results were obtained with [Q(3)A(4)Y(15)L(16)]-IGF-I. Cross-linking experiments using iodinated or unlabeled IGFBP-3 and anti-IGF-IR antibodies indicated that the inhibitory effects do not involve direct interaction between IGFBP-3 and IGF-IR. The inhibition appeared to be specific to IGFBP-3, because IGFBP-1 and IGFBP-5 at 10 nM had no significant effect. Also, inhibition was restricted to the IGF receptor, because IGFBP-3 failed to inhibit the tyrosine kinase activity of the insulin receptor stimulated by physiological concentrations of insulin. Our results provide the first demonstration that IGFBP-3 can specifically modulate the IGF-I signaling pathway independently of its IGF-I-binding ability. They also reveal a regulatory mechanism specific to the type 1 IGF receptor, with no effect on insulin receptor activation.
Phosphatidylinositol 3-kinase in myogenesis.
Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc.
Quick links (PubMed)
- PMID 8930132 — 1996 · Des(1-3)IGF-I: a truncated form of insulin-like growth factor-I.
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- PMID 1715381 — 1991 · The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)…
- PMID 1883485 — 1991 · Expression, purification and characterization of secreted recombinant hu…
- PMID 7779409 — 1994 · Des (1-3) IGF-I potently enhances differentiated cell growth in olfactor…
- PMID 2280209 — 1990 · Enhanced potency of truncated insulin-like growth factor-I (des(1-3)IGF-…
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- PMID 1928375 — 1991 · IGF-I and its variant, des-(1-3)IGF-I, enhance growth in rats with reduc…
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- PMID 2539101 — 1989 · Insulin-like growth factor (IGF)-binding proteins inhibit the biological…
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- PMID 10959407 — 2000 · Involvement of growth factors in the regulation of pubertal mammary grow…
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- PMID 8940346 — 1996 · Combination of insulin-like growth factor (IGF)-I and IGF-binding protei…
- PMID 9389551 — 1997 · The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits…
- PMID 11735239 — 2001 · Effect of IGFBP-3 on IGF- and IGF-analogue-induced insulin-like growth f…
- PMID 1388667 — 1992 · An evaluation of different enzymatic cleavage methods for recombinant fu…
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- PMID 1371669 — 1992 · Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are a…
- PMID 1378742 — 1992 · Novel recombinant fusion protein analogues of insulin-like growth factor…
- PMID 11025411 — 2000 · Interactions of IGF-1 with the blood-brain barrier in vivo and in situ.
- PMID 11145572 — 2001 · Insulin-like growth factor (IGF) binding protein-3 inhibits type 1 IGF r…
- PMID 21235885 — 1997 · Phosphatidylinositol 3-kinase in myogenesis.